Papillary thyroid cancers (PTC) are associated with a high prevalence of non-overlapping mutations of RET, TRK, RAS, and BRAF. As these signaling proteins form part of the classical MAPK pathway, this has been interpreted as evidence for a critical role of MAPK activation in initiation and maintenance of thyroid cancer. However thyroid cancers are also associated with mutations of genes encoding effectors signaling along the phosphatidylinositol 3-kinase (PI3K) pathway, yet whether constitutive activation of PI3K-AKT cooperates or not in certain thyroid tumors with MAPK to drive the tumorigenic process is unknown. RAS oncoproteins signal through both pathways, whereas oncogenic BRAF signals preferentially through MAPK, providing a useful paradigm in which to explore these interactions in mouse cancer models. The intensity of the oncogenic signal is an important variable, and we intend to look at these interactions through genetic manipulations that approximate pathophysiological conditions. For this we have generated mice with a conditionally gene-targeted activating mutation of H-ras, and obtained an analogous model for B-raf. 1) We will explore whether these oncoproteins initiate cell transformation through induction of a DNA damage response, followed by senescence. 2) We will expand on our observation that a thyroid-specific knock-in of H-ras markedly increases mutation frequency in mice, by using mouse reporter strains to test for different mutation types. 3) We will then examine whether H-ras or B-raf activation cooperate with loss of the tumor suppressor Pten to promote thyroid tumorigenesis in different genetic backgrounds, and explore the mechanisms involved. 4) Finally, we will test the hypothesis that genetic determinants of MAPK and PI3K activation in a large panel of suitably genotyped human thyroid cancer cell lines predicts responsiveness to pathway inhibitors. Public Health Relevance Statement: In this proposal, we intend to use mouse models to reconstruct the very first stages of thyroid cancer development. The very first genetic changes that get the tumor started are particularly important, because cancers remain dependent on their abnormal activity even though they continue to accumulate damage of other genes over time. This will help us decide what key genetic abnormalities to focus on as we develop specific treatments for patients with the most advanced forms of the disease.